...multiple) rAAV-CAG::FLEX-rev:: hM4D-2a-GFP AAV1 Chemogenetics Scott Sternson New viral prep pAAV-CaMKIIa-hM3D...mCherry AAV1 Chemogenetics Bryan Roth New serotype pAAV-CaMKIIa-hM4D(Gi)-mCherry AAV1 Chemogenetics Bryan ...mCherry AAV1 Chemogenetics Bryan Roth New serotype pAAV-hSyn-hM4D(Gi)-mCherry AAV1 Chemogenetics Bryan Roth...AAV-PHPeB Optogenetics Jonathan Ting New viral prep AiP14033: pAAV-AiE0779m_3xC2-minBG-CoChR-EGFP-WPRE3-...AAV-PHPeB Optogenetics Jonathan Ting New viral prep AiP14035: pAAV-AiE0452h_3xC2-minBG-CoChR-EGFP-WPRE3-...pAAV-AiE0873m_3xC2-minBG-CoChR-EGFP-WPRE3-BGHpA (Alias: CN5140) AAV-PHPeB Optogenetics Jonathan Ting New viral... Brian O'Neill.  More resources on the Addgene blog Addgene's Data Hub What is a Virus (and a Viral Vector...

...highlighted back in 2015, where sdAbs were used to create GFP scaffolds for transcriptional activation.  sdAbs ...from addgene.org  Plasmids encoding nanobodies at Addgene Sybody Generation Toolbox from Addgene     ...only antibodies. (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable ...The UN General Assembly has declared 2024 the International Year of Camelids. The declaration is intended...to human populations around the world. Here at Addgene, we love camelids too, though not just for the ...chromobodies,” which are single-domain antibodies genetically fused to fluorescent proteins and used to visualize... synthetic single-domain antibody libraries to generate new sdAbs. There has also been some interesting...

... Fig. 3: Image of Addgene’s western blot for the myc-tagged protein GFP-smFP-myc expressed from ...C-terminal region of the human proto-oncogene c-Myc. Since the generation of the first anti-c-Myc [9E10] (...high-throughput screening platform to apply saturating mutagenesis on 21 positions in the protein sequence of the...approach and outcomes of the screen for improved genetically-encoded voltage indicators (GEVIs) based on the...kinetics. They demonstrate the use of HcKCR1 as an optogenetic silencer in experiments with mouse cortical neurons... HcKCR2, making it more suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective...control K+ gradients and can be used as a potent optogenetic silencer. The current work also proposes promising...

...contains circularly permuted green fluorescent protein (GFP), and nLightR uses a red fluorescent protein, mApple...Imaging Tools Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent... visualize their activity. In 2019, the first generation of norepinephrine sensors was developed, named...norepinephrine (Feng et al., 2019). But this first generation of tools had limited sensitivity and speed and... models of nLightG (left) and nLightR (right) generated using ColabFold (Mirdita et al., 2022). B, Representative...Speed One of the major advantages of this next generation of norepinephrine sensors is their speed. nLightG...GRABNE were more sensitive than nLightR, both generating a measurable signal after electrical stimulation...

...targeting a gene associated with diphtheria toxin lethality and were sorted into single wells to generate clonal...instance, it’s much more difficult to introduce a gene of interest into primary cells, so most primary ...Neuronal stem cells transduced with iTOP resulting in GFP expression. Image courtesy of Diego D'Astolfo. ... risk of viral integration causing mutations and gene expression changes elsewhere in the genome. Second...j.cell.2015.03.028. PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters Previous...

...collection is now complete! pAAV-CAG-GFP (plasmid 37825) and pAAV-hSyn-EGFP (plasmid 50465) are now available...include: EGFP-expressing AAV for serotype testing Calcium sensors and GABA sensors Optogenetics Recombinases... pAAV.hSynap.iGABASnFR (112159-AAV1) Optogenetics Optogenetics continues to be a useful method for manipulating...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols... testing AAV, which are small (20 ul) samples of EGFP-expressing AAV packaged in various serotypes. This...recently released these controls: pAAV-hSyn-DIO-EGFP (50457-AAV5): The Synapsin promoter directs broad...broad, neuronal expression. AAV pCAG-FLEX-EGFP-WPRE (51502-AAV5): The CAG promoter directs ubiquitous expression...

...destination vector contains GFP, enabling you to select cells with high GFP expression. These cells have...sequence between two target sites. Modifying multiple genes at once. Using multiplexed gRNAs can target multiple...simultaneous multiplex editing of six different genes. This system doesn’t rely on any additional or host-dependent...from this paper at Addgene. 9. Yin, Linlin, et al. “Multiplex Conditional Mutagenesis Using Transgenic ...plasmids from this paper at Addgene. Additional Resources on the Addgene Blog Browse Our CRISPR Topic...add multiple gRNAs on one plasmid. Sound good? Addgene has many tools to help you multiplex - we’ll use...multiplex gRNAs, some being: Using dual nickases to generate a knockout or edit. This can help reduce off-target...

...plasmids from this publication at Addgene. Multichromatic control of gene expression in Escherichia coli...optimized by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust. Synthetic...used to engineer new pathways with sophisticated gene expression controls. Most cell types do not respond... can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive two-component...via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly... was only about 10-fold, precluding the study of gene expression at very high or very low levels. Schmidl...researchers to better fine-tune the level of output gene expression based on the input light intensity. In...

...test for your gene of interest may be another method to assess transfection success. GFP is often used...lines and are not used for general plasmid selection. Instead, reporter genes are often employed to easily...conjunction with positive selection to ensure your gene gets targeted to a specific location within the ...used selection markers are listed below: Name Gene Conferring Resistance Cell Types* Mode of Action...use G418. This can be confusing since the neo/kan gene confers G418 resistance; however, like gentamicin...Read More: Plasmids 101: Antibiotic Resistance Genes Plasmids 101: Origin of Replication All Plasmids...the genetic traits of the foreign material and will subsequently pass it on to future generations. Stably...

... for gene expression, turning transcription on and off by binding proximal to their target genes. Since...Since gene expression patterns determine everything from stem cell differentiation fate to tumor suppression...library comes either with or without mCherry and GFP fluorescent controls. Alternatively, you can order...from the library to further investigate specific genes and TF isoforms. Due to the size of the collection...use to generate the relevant disease model. Accurate modeling of a disease depends on both genetic and expression... not yet available, just ask an Addgene scientist at help@addgene.org! References and Resources  References...scientific community. Recently, the Zhang lab generated a TF library (aka the MORF Collection) and an ...

...that control cell cycle gene expression.  Additional Resources on the Addgene Blog Visit the fluorescent...page Get the basics on GFP Find microbiology blog posts Resources on Addgene.org Find fluorescent protein...work in Lin Tian’s lab at UC Davis is to generate a genetically encoded fluorescent sensor for serotonin...sequence an individual’s genome. But when they find genetic variants, what does that mean? Previous research...of Washington sought to address the effects of genetic variants in a high throughput manner. She helped...variant library is monitored by fusing the variants to eGFP followed by an internal ribosomal entry site and...a variant has wild-type levels of abundance, the eGFP will be stable and fluoresce. But if the variant...

... also includes a GFP construct as a control. For easier portability, all viral genes are flanked by EcoRI...coronavirus or COVID-19 research, please email Addgene at help@addgene.org with your order number and so it can .... Sign up for email alerts on the Krogan lab's Addgene page to get notified!) Attacking coronavirus, one...reasons, the QCRG is delighted to be partnering with Addgene, who will be taking over the distribution to expand... coronavirus related plasmids and resources at Addgene* Many thanks to our guest bloggers Manon Eckhardt...

...developed four acoustic reporter gene (ARG) plasmids to better monitor gene expression in vivo using ultrasound...non-immunogenic violet-excited GFP variant Vex. To understand the impact of this gene editing tool on the immune...plasmids at Addgene. Liu Q et al., Neuron. 2019. PubMed PMID: 30704911. Acoustic reporter genes for noninvasive...by Lukas Morgan  Listen to the acoustic reporter gene podcast segment In recent years, scientists are ...through a host organism, the expression of some genes can change depending on the location. Current methods...of microbes rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue...ultrasound. These ARG plasmids contain genes for “gas vesicles,” a gas-filled protein structure that water-bound...

...They could also switch from FLAG to GFP or a different epitope. Addgene: Do you have any additional tips,...homology arm as a gBlocks® Gene Fragment from Integrated DNA technologies. Generally speaking, anyone using... an effort to track down every last one of those gene regulatory proteins and the places in the genome...together on several projects and this is one related to gene expression. One goal of the ENCODE project is to...with detailed protocols, go to www.addgene.org/crispr/tagging. Addgene: What motivated you to adapt CRISPR...tagging system, which they’ve now made available at Addgene. For more details on the plasmids that are currently...can now look at many more transcription factors. Addgene: How does the system work? Mendenhall: The system...

...into contact with than GFP. Similarly, if the viral vector carries an shRNA to a gene essential for cellular...looks like the genetic cargo in one plasmid (the transfer plasmid), the envelope (Env) gene on a separate...with those particles. Since the Env, Gag, and Pol genes are not encoded in the transfer plasmid, the component...Resources Resources on Addgene.org Addgene viral services Addgene Biosafety Guide Addgene Viral Plasmids ...? That’s okay! In this article we will discuss general viral safety considerations and best practices ...of what is being integrated by the virus. If an oncogene overexpression construct is being delivered, that... and the same holds true for types of viruses generated in the lab. The four most common types of viruses...

...ones? Many of the available biosensors use a single GFP or CFP/YFP-derived FRET pair. If you need to free...Resources on Addgene.org Addgene’s AAV Guide Fluorescent Proteins: Biosensors Addgene’s Viral Services...calcium sensors. Selecting a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent...neuronal cell body or the axon (GCaMP6 only). The genetic elements required for Drosophila studies have been...chemical biology. 2021 May;17(5):509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain...Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded fluorescent indicators for organellar...SA, Looger LL. Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harbor...

...line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor, Hoechst, and overlay).   ...(DKFZ) who has developed new redox sensitive GFP2 (roGFP2)-based biosensors for live cells. His work focuses..., Pedre Pérez’s biosensor fuses MPST to roGFP2. Because roGFP2 contains two cysteines near the chromophore...developed a dual virus system using both optogenetics and chemogenetics to study the activity of parvalbumin...in the lab. He has recently developed a third-generation intensiometric glutamate sensing fluorescent ...catalyzes the desulfuration of 3-mercaptopyruvate and generates an enzyme-bound hydropersulfide that is then transferred...the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols and change its fluorescence properties...

...Cre-lox Control Protein Activity with GFP Resources at Addgene.org Find KTR reporter plasmids Find photoactivatable...spatiotemporal resolution than pharmacological or genetic approaches, but only a few such methods exist and...Solution: In Zhou et al, the Lin lab present a generic design for creating photoswitchable kinases and...possible to create other photoswitchable kinases? The general design of psMEK1 also worked for creating three...PMCID: PMC4097317. Additional Resources on the Addgene Blog Learn More about Photoactivatable Fluorescent...

...excised by Cre recombinase. Addgene has several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression...investigation into the regulation of your favorite gene. But have no fear - I’m here to introduce you to...genome – could be affected, leading to unpredicted gene expression changes (Sapozhnikov and Szyf 2021, Charlton...interact with transcriptional machinery to modify local gene expression, confounding any potential conclusions...causal relationship between DNA demethylation and gene expression (Sapozhnikov and Szyf 2021).    ... expressing blasticidin or puromycin resistance genes.  It’s that simple! Good luck and happy editing...Vogelstein B (1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts...

... enriched relative to controls using Split-BioID-GFP and BioID run on six unrelated proteins. One caveat...study binary protein interactions. However, FRET generally only tells you if two proteins interact and cannot...with your research? Plasmids are available from Addgene! References Schopp, I.M., Ramirez, C.C., Debeljak...Central PMCID: 5053326. Additional Resources on the Addgene Blog Learn about techniques for visualizing protein...-DNA Interactions with CUT&RUN Resources at Addgene.org Find plasmids from the Bethune Lab Find Plasmids...